1. The Relationship between Single-Channel and Whole-Cell Conductance in the T-type Ca2+ Channel CaV3.1 
Katie C. Bittner, Dorothy A. Hanck 
Biophysical Journal 
Volume 95, Issue 2, Pages (July 2008)
DOI: /biophysj
Copyright © 2008 The Biophysical Society Terms and Conditions

2. FIGURE 1
(A) Family of representative whole-cell currents from the same cell in the presence of 5mMBa2+ (middle) and 5mMCa2+ (lower) in response to 5-mV voltage steps from a holding potential of −110mV over the range −90–+40mV. Cells were depolarized once every 5s to ensure full recovery from inactivation. (B) Average ± SE of the current-voltage relationship in the presence of 5mMBa2+ (circles) and Ca2+ (squares) for five cells studied in the same way. Currents were normalized to the peak Ca2+ current in each cell.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

3. FIGURE 2
Representative single T-type Ca2+ channel currents and ensemble averages. Capacity- and leak-corrected single-channel currents and the corresponding ensemble currents from a single cell-attached patch elicited with step depolarizations to the indicated potentials (left) in the presence of 20mMCa2+. Currents shown include 15ms at the holding potential (−100mV) and 100ms at the depolarized potential. Currents were sampled at 20kHz, filtered at 2kHz, and then downsampled to 4kHz offline. For graphical purposes, traces were additionally filtered by adjacently averaging by 3. The seal resistance of the patch was >100GΩ.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

4. FIGURE 3
Measured current amplitudes are the same when currents are elicited using depolarizations or tail-current protocols. (A) Capacity- and leak-corrected single-channel currents from a cell-attached patch elicited with a 50-mV step depolarization (−100mV to −50mV) with 20mMCa2+ as the permeant ion. Data were sampled at 20kHz, filtered at 2kHz, and downsampled to 4kHz offline. (B) Capacity- and leak-corrected single-channel currents from the same patch as in A elicited with the tail-current protocol shown above. Patches were depolarized to 0mV for 3ms and subsequently hyperpolarized for 80ms, in this case to −50mV. Single-channel current amplitudes were determined from all-points histograms from selected segments of active traces. Selected segments were marked (dotted lines) from ∼100 sweeps per potential, concatenated, and used to generate the amplitude histogram. The data were binned into 30-fA bins and fit with a sum of two Gaussians. The signal/noise ratio was ∼3 at −30mV and increased to ∼7 at −50mV. (C) Amplitude histogram for currents elicited with 50-mV step depolarizations. The dotted black lines are the Gaussian fit of the closed and open states, and the solid line is the sum of the Gaussians. The open-channel current at −50mV was measured to be −0.27pA. The area under the curve bears no relationship to open and closed durations, because the data used to generate the amplitude histogram excluded much of the closed-state data. (D) The current-voltage relationship for the patch shown in A and B. The solid line is a linear fit to the data with a slope conductance of 5.9±0.8pS. The two points at −50mV were calculated using the protocols from A and B.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

5. FIGURE 4
Nonlinearity of the whole-cell and single-channel current-voltage relationships at positive potentials. (A) Family of capacity- and leak-corrected representative currents recorded during steps between −150mV and 130mV immediately after a 1-ms depolarization to 0mV from a holding potential of −100mV. Sweeps are shown for every 20mV. (B) Representative whole-cell open-channel I/V relationship for the cell in A. For whole-cell experiments, the bath solution was the same as the 20-mM-Ca2+ single-channel pipette solution, and the pipette solution contained (in mM)140KCl, 1CaCl2, 10EGTA, 5MgCl2, and 10HEPES, titrated to pH7.4 with KOH. Data were filtered at 100kHz by an 8-pole lowpass Bessel filter and digitized at 200kHz. Although the currents are rather large, the time to peak of the tail currents was within the same range as that of currents of much smaller amplitude (0.15ms) indicating that voltage control was maintained (data not shown). (C) Single-channel current-voltage relationship. Currents shown are from eight different patches in 20mMCa2+. Tail protocols were used to elicit currents from −70 to −120mV and 100-ms step depolarizations were used from 0 to −70mV, where each symbol represents an individual determination from a single patch. Single-channel conductance was highly sensitive to the voltage range over which it is measured. The dotted line is a linear fit to the data between −20mV and −60mV (γ=4.2±0.3pS) (solid squares). The solid line is a linear fit of the data between −60mV and −120mV (γ=8.4±0.8pS) (solid circles).
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

6. FIGURE 5
Representative single-channel Ba2+ currents. (A) Voltage protocol used to elicit single-channel currents in B and C. Patches were held at −100mV, depolarized to 0mV for 3ms, and then hyperpolarized to the test potential. Recordings were made during the final 15ms of the 3-s interpulse interval, during the 3-ms depolarization to 0mV, and for 80ms at the test potential. (B) Capacity- and leak-corrected single-channel currents elicited with the tail protocol shown in A from a holding potential of −100mV in 5mMBa2+ (upper) and 60mMBa2+ (lower). Top currents were sampled at 4kHz and filtered at 2kHz. Bottom currents were sampled at 20kHz and filtered at 2kHz and then downsampled to 4kHz offline. The seal resistance was >100GΩ in each patch.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

7. FIGURE 6
(A) Single-channel currents as a function of potential measured using an amplitude histogram (open circles, n=4) or QuB (solid squares, n=5), as described in text, using the diagram of the model shown. (B) Current-voltage relationship for both the main conductance state (solid circles) and subconductance state (solid squares) obtained using QuB in the presence of 40mMCa2+ (n=5). The solid line is a linear fit of the main-state data with a slope conductance of 9.0±0.6pS. The dotted line is a linear fit of the subconductance-state data with a slope conductance of 6.0±1.0pS. (Inset) Idealized currents in the presence of 40mMCa2+. Currents were sampled at 20kHz, filtered at 2kHz, and downsampled offline to 4kHz. (C) The current-voltage relationship for both the main conductance state (open circles) and subconductance state (open squares) obtained using QuB in the presence of 115mMCa2+ (n=4). The solid line is a linear fit of the main-state data with a slope conductance of 9.4±1.5pS. The dotted line is a linear fit of the subconductance-state data with a slope conductance of 6.8±0.9pS. (Inset) Idealized currents in the presence of 115mMCa2+. Currents were sampled at 20kHz, filtered at 2kHz, and downsampled offline to 4kHz. (D, left) The relative fractional occupancy of the subconductance states for 40mMCa2+ (solid squares) and 115mMCa2+ (open circles). Fractional occupancy was calculated as (occupancy of the subconductance state)/(occupancy of the subconductance state + occupancy of the main conductance state). (D, right) The grouped relative fractional occupancy of the subconductance state for 40mMCa2+ (n=5) and 115mMCa2+ (n=4). The data from each cell over a voltage range of −60mV to −80mV were averaged and the values from each cell were averaged and displayed as mean±SE. *—Statistical significance as measured by a Student t-test (p=0.03).
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

8. FIGURE 7
Divalent ion concentration dependence of single-channel conductance. Slope conductance over a range of Ca2+ concentrations (squares, n=19) and Ba2+ concentrations (circles, n=26) (5–115mM). All of the conductance measurements were made using all-points histograms except 115mMCa2+. Conductance was determined by the linear regression to the currents from multiple patches at each concentration. Data are displayed as the estimate±SE. For each concentration, there were at least three patches with current amplitudes at three potentials, except in two cases: for 5mMBa2+, there was only one patch with three potentials and in 5mMCa2+ there were only two patches with three potentials. The data are fit with a Langmuir isotherm function. For Ca2+, γmax=9.5±0.4pS and KM=2.3±0.7mM. For Ba2+, γmax=10.3±0.5pS; KM=7.9±1.3mM. (Inset) The concentration dependence is displayed on a semilogarithmic scale.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

9. FIGURE 8
(A) Whole-cell current-voltage relationship in the presence of 5mM divalent (same as Fig. 1 B) and I/V voltage protocol (inset). (B) The whole-cell current-voltage relationship in the presence of 60mMCa2+ (solid squares) and Ba2+ (open circles). The data are displayed as mean ± SE normalized to the peak current in Ca2+. In the cases where the error bars are not visible, it is because they are smaller than the size of the symbol. For whole-cell experiments, the bath solution was the same as the single-channel pipette solution containing 60mMCa2+ and 60mMBa2+, respectively. (C) Average whole-cell open-channel I/V relationship in 5mMCa2+ (solid squares) and 5mMBa2+ (open circles) and voltage protocol (inset). The data are displayed as mean±SE normalized to the current in Ca2+ at −80mV. In the cases where the error bars are not visible, it is because they are smaller than the size of the symbol. The half-solid circles are the Ba2+ data shifted 10mV positive (as described in text). Data were filtered at 100kHz by an 8-pole low-pass Bessel filter and digitized at 200kHz. Currents were recorded at hyperpolarized potentials after depolarization to +60mV for 1ms. (Inset) Capacity- and leak-corrected representative currents in 5mMBa2+ (upper) and Ca2+ (lower). Traces were filtered offline to 20kHz and the first 0.05ms are not shown. (D) Average whole-cell open-channel I/V relationship in 60mMCa2+ (solid squares) and 60mMBa2+ (open circles). The data are displayed as the mean±SE normalized to the Ca2+ at −80mV. In the cases where the error bars are not visible, it is because they are smaller than the size of the symbol. The half-solid circles are the Ba2+ data shifted by 20mV (as described in text). Currents were recorded at hyperpolarized potentials after a brief depolarization to +115mV for 1ms. Data were filtered at 100kHz by an 8-pole low-pass Bessel filter and digitized at 200kHz. (Inset) Capacity- and leak-corrected representative currents in 60mMBa2+ (upper) and Ca2+ (lower). Traces were filtered offline to 20kHz and the first 0.06ms are not shown.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions